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. 2019 Jan 11;68(10):1751–1763. doi: 10.1136/gutjnl-2017-315318

Figure 6.

Figure 6

FOXD1/FOXJ1 axis inhibits nuclear factor  kappa B (NF-κB) pathway and CDX2 transcription. (A) Gastric epithelial cell line (GES)-1 cells infected with FOXJ1 or FOXD1 short hairpin RNA (shRNA) were transfected with a pGL4.32 vector containing NF-κB-luciferase reporter along with FOXJ1-expressing vectors. NF-κB-luciferase activity was then measured by using GloMax plate reader. (B) GES-1 cells were infected with FOXJ1 or FOXD1 shRNA, followed by transfection of FOXJ1-expressing vectors or control and expression of NF-κB-pathway-regulated genes were examined by qRT-PCR. GAPDH RNA was used as internal control. (C) Left: the representative images of IHC staining for p-p65 and CDX2 in normal tissues and intestinal metaplasia (IM) tissues. Scale bars: 100 µm; 500 µm (insets). Right: association between expression of p-p65 and CDX2 levels in IM specimens. (D) Serially truncated CDX2 promoter constructs were cloned to pGL3-luciferase reporter plasmids and transfected into GES-1 cells. Four hours after transfection, cells were treated with CDCA (100 µM) for 24 hours and the relative luciferase activities were determined 72 hours after ending of CDCA treatment. (E) A Ch-IP assay demonstrated the direct binding of p65 to the CDX2 promoter in GES-1 cells. M: Marker. (F) qRT-PCR of the Ch-IP products validated the binding capacity of p65 to the CDX2 promoter. Means±SEM of a representative experiment (n=3) performed in triplicates are shown. *P<0.05; **p<0.01; ***p<0.001. N.S., not significant.